This invention relates to fungal/bacterial antagonist combinations, a seed coated with said combinations and a plant protected from plant pathogens by said combinations. In particular, the invention relates to fungal/bacterial antagonist combinations and their use for controlling plant pathogens.
Early and late season stalk and root rot are major causes of crop loss. A variety of plants are affected, including tomatoes, peppers, turf grass, soybeans, sunflower, wheat and corn. The pathogens that cause these symptoms include fungi of the genera Fusarium, Phythium, Phytophthora and Penicillium. 
One approach to solving the problem of early season damping off of plants is treatment of seeds with fungicides, such as captan, metalaxyl and Maxim. Although these chemicals enhance seed germination and seedling stand by inhibiting the pathogenic ability of Phythium spp. (active in cool, wet soils), they have no activity against the pathogenic fungi that are responsible for late season root and stalk rot.
Fusarium and Penicillium are the pathogens responsible for late season root and stalk rot. These pathogens prefer the warm, dry conditions that occur late in the growing season. There is no chemical or biological fungicide available that addresses the problem of late season root and stalk rot in corn. Currently, the only way to deal with this problem is to periodically rotate to a non-susceptible crop to reduce pathogen numbers. Corn growers can also select hybrids that have better “standability,” but such hybrids usually have lower yields. Unfortunately, the corn varieties with the highest yields are usually those most susceptible to late season root and stalk rot.
Trichoderma is a genus of fungi that contains about 20 species. Synonyms for the genus name include Aleurisma and Sporoderma. Trichoderma virens, which is also called Gliocladium virens, is a member of the genus. The natural habitats of these fungi include soil and plant material. A member of the genus, Trichoderma harzianum KRL-AG2 (ATCC 20847) also known as strain T-22, is used as a biocontrol agent that is applied as a seed or soil treatment or on cuttings and transplants. Strains of the species, Trichoderma virens, have also been used for control of damping off diseases in plants. For example, Trichoderma (Gliocladium) virens G1-21 is known and commercially available at a reasonable price, and is being marketed under the trademark SoilGuard® 12G (EPA Registration Number: 70051-3 and EPA Establishment Number: 067250-IL-001). It is manufactured by Thermo Trilogy Corporation of Columbia, Md. Other known and commercially available Trichoderma virens strains include those having the following ATCC accession numbers: 10043, 10044, 10045, 13213, 13362, 204067, 204443, 204444, 204445, 20903, 20904, 20906, 24290, 42955, 44327, 44734, 48179, 52045, 52199, 58676, 58677, 58678, 62399, 64271, 74180, 9645, MYA-297, MYA-298, MYA-649 and MYA-650.
Bacillus is a genus of rod-shaped, gram-positive, aerobic or (under some conditions) anaerobic bacteria. Bacillus species are widely found in soil and water and some have been used to control plant diseases, including root rot. Bacillus amyloliquefaciens is a spore-forming member of the genus. Bacillus amyloliquefaciens L.L. Campbell strain F (ATCC 23350) is the type strain for the species. Other known and commercially available Bacillus amyloliquefaciens strains include those having the following ATCC accession numbers: 23842, 23843, 23844, 23845, 31592, 49763, 53495 and BAA-390 (Int. J. Sys. Bacteriol. 37:69-71, 1987; J. Bacteriol. 94:1124-1130, 1967).
Bacillus amyloliquefaciens is also called Bacillus subtilis var. amyloliquefaciens by some investigators. A protease produced from Bacillus subtilis var. amyloliquefaciens is commonly used as a tenderized for raw meat products. According to the U.S. Environmental Protection Agency (EPA), Bacillus subtilis var. amyloliquefaciens strain FZB24 is a naturally-occurring microorganism and widespread in the environment. Bacillus subtilis var. amyloliquefaciens FZB24 (EPA Registration Number: 72098-5 and EPA Establishment Number: 73386-DEU-001) is known and commercially available at a reasonable price, being marketed under the trademark Taegro® by Earth Bioscience, Inc. of Fairfield, Conn.
Bacillus lentimorbus is another spore-forming member of the genus. Bacillus lentimorbus Dutky 1940 (ATCC 14707) is the type strain for the species (Skerman, V. B. D., McGowan, V., and Sneath, P. H. A., Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30: 225-420, 1980). Some researchers consider Bacillus lentimorbus to be a variety of Bacillus popilliae. Bacillus lentimorbus and Bacillus popilliae have recently been reclassified as Paenibacillus lentimorbus and Paenibacillus popilliae (Pettersson, B., Rippere, K. E., Yousten, A. A. and Priest, F. G., Transfer of Bacillus lentimorbus and Bacillus popilliae to the genus Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov., Int. J. Syst. Bacteriol. 49: 531-540, 1999). Bacillus lentimorbus and Bacillus popilliae are the causative agents of milky disease in Japanese beetles and related scarab larvae and “milky spore” powders are sold under the trade names, “Doom,” “Milky Spore,” “Japidemic” “Grub Killer” and “Grub Attack,” for biocontrol of these insects.
Background art biocontrol products have comprised the bacterium Burkholderia cepacia, which is also known as Pseudomonas cepacia. This bacterium has been implicated as a human pathogen. Furthermore, it has little or no shelf life unless refrigerated at 4 degrees Centigrade at a minimum of 20 percent moisture.
The background art is characterized by U.S. Pat. Nos. 4,476,881; 4,489,161; 4,642,131; 4,668,512; 4,678,669; 4,713,342; 4,724,147; 4,748,021; 4,818,530; 4,828,600; 4,877,738; 4,915,944; 4,952,229; 5,047,239; 5,049,379; 5,071,462; 5,068,105; 5,084,272; 5,194,258; 5,238,690; 5,260,213; 5,266,316; 5,273,749; 5,300,127; 5,344,647; 5,401,655; 5,422,107; 5,455,028; 5,409,509; 5,552,138; 5,589,381; 5,614,188; 5,628,144; 5,632,987; 5,645,831; 5,665,354; 5,667,779; 5,695,982; 5,702,701; 5,753,222; 5,852,054; 5,869,042; 5,882,641; 5,882,915; 5,906,818; 5,916,029; 5,919,447; 5,922,603; 5,972,689; 5,974,734; 5,994,117; 5,998,196; 6,015,553; 6,017,525; 6,030,610; 6,033,659; 6,060,051; and 6,103,228.
Gravely et al. in U.S. Pat. No. 4,476,881 disclose a mixed complementary culture of bacteria and fungi that is used to degrade pectin and cellulose components of tobacco materials. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Papavizas in U.S. Pat. No. 4,489,161 discloses a strain of the fungus Trichoderma viride that is an effective biocontrol agent for Fusarium wilt of chrysanthemum. The invention is limited in that it teaches use of a different microorganism.
Hoitinik in U.S. Pat. No. 4,642,131 discloses a process for production of a disease-suppressive compost and a microorganism culture for use therein. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Lewis et al. in U.S. Pat. No. 4,668,512 disclose a method for preparing pellets containing living biocontrol fungi and nutrients. The invention is limited in that it teaches a process that involves use of different microorganisms.
Ricard in U.S. Pat. No. 4,678,669 discloses a method of using immunizing commensals to control soil-borne pathogens. The invention is limited in that it teaches use of different microorganisms.
Chet et al. in U.S. Pat. No. 4,713,342 disclose a novel isolate of Trichoderma and it use. The invention is limited in that it teaches use of a different microorganism.
Marois et al in U.S. Pat. No. 4,724,147 disclose a method for preparing pellets containing living biocontrol fungi. The invention is limited in that it teaches a process that involves use of different microorganisms.
Chet et al. in U.S. Pat. No. 4,748,021 disclose antifungal compositions containing Trichoderma active against Fusarium. The invention is limited in that it teaches use of a different microorganism.
Marois et al. in U.S. Pat. No. 4,818,530 disclose a method for preparing pellets containing living biocontrol fungi. The invention is limited in that it teaches a process that involves use of different microorganisms.
McCabe et al. in U.S. Pat. No. 4,828,600 disclose a biological inoculant for corn. The invention is limited in that it teaches use of different microorganisms.
Handelsman et al. in U.S. Pat. No. 4,877,738 disclose a new microorganism culture and a method for biological control of damping off and root rot. The invention is limited in that it teaches a process that involves use of a different microorganism, Bacillus cereus. 
Chet et al. in U.S. Pat. No. 4,915,944 disclose a novel isolate of Trichoderma and fungicidal compositions containing it. The invention is limited in that it teaches use of a different microorganism.
Muir in U.S. Pat. No. 4,952,229 discloses a soil and foliar supplement. The invention is limited in that it teaches use of different microorganisms.
Pusey in U.S. Pat. No. 5,047,239 discloses a biological control agent for fruit rot. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Handelsman et al. in U.S. Pat. No. 5,049,379 disclose a fungicidal toxin and a method and an inoculum for controlling root rot and damping off. The invention is limited in that it teaches use of a different microorganism, Bacillus cereus. 
Kimura in U.S. Pat. No. 5,071,462 discloses a method and apparatus for producing an organic fertilizer. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Lewis et al. in U.S. Pat. No. 5,068,105 disclose a fungal formulation for biocontrol of soil-borne plant pathogens. The invention is limited in that it teaches use of different microorganisms.
Speakman et al. in U.S. Pat. No. 5,084,272 disclose a Trichoderma fungus and a fungicide that contains it. The invention is limited in that it teaches use of a different microorganism.
Pauu et al. in U.S. Pat. No. 5,194,258 disclose a method for producing enhanced biocontrol agents. The invention is limited in that it teaches use of different microorganisms.
Elad et al. in U.S. Pat. No. 5,238,690 disclose a novel Trichoderma culture and biological compositions containing it. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Harman et al. in U.S. Pat. No. 5,260,213 disclose fused biocontrol agents. The invention is limited in that it teaches use of different microorganisms.
Elad et al. in U.S. Pat. No. 5,266,316 disclose a novel isolate of Trichoderma harzianum and fungicidal compositions containing it. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Bok et al. in U.S. Pat. No. 5,273,749 disclose a process for preparing coated microbial pesticides and the products of the process. The invention is limited in that it teaches use of different microorganisms.
Williams in U.S. Pat. No. 5,300,127 discloses seed coatings. The invention is limited in that it teaches use of different microorganisms.
Rossall in U.S. Pat. No. 5,344,647 discloses a Bacillus strain that has antimicrobial activity. The invention is limited in that it teaches use of different microorganisms.
Kijima et al. in U.S. Pat. No. 5,401,655 disclose a process for biologically preventing plant diseases. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Kubota in U.S. Pat. No. 5,422,107 discloses a novel fungus and a fungicide containing it. The invention is limited in that it teaches use of a different microorganism.
O'Donnell in U.S. Pat. No. 5,455,028 discloses a method of inhibiting fungi. The invention is limited in that it teaches use of a different microorganism.
Burth et al. in U.S. Pat. No. 5,409,509 disclose a seed treatment. The invention is limited in that it teaches use of different microorganisms.
Shanmuganathan in U.S. Pat. No. 5,525,132 discloses “compositions for the treatment/prevention of microbial diseases of fruit comprising as effective amount of at least one yeast strain . . . ” (Abstract). In Example 2, strains are tested separately and are not combined. The disclosed strain D9 was effective and Trichoderma viridie was not. Neither was Bacillus subtilis effective. The reference teaches that “it is very unpredictable whether a particular yeast species would be effective in the treatment/prevention of microbial disease in fruit” (col. 2, lines 26-28).
Handelsman et al. in U.S. Pat. No. 5,552,138 disclose a novel strain of Bacillus cerus and a method of protecting plants with the strain. The invention is limited in that it teaches use of a different microorganism.
Neyra et al. in U.S. Pat. No. 5,589,381 disclose a novel strain of Bacillus licheniformis that produces an antifungal agent and a use for the strain. The invention is limited in that it teaches use of a different microorganism.
Urano et al. in U.S. Pat. No. 5,614,188 disclose an anti-Fusarium composition containing strains of Bacillus sp. The invention is limited in that it teaches use of different microorganisms.
Eastin in U.S. Pat. No. 5,628,144 discloses solid matrix priming of seeds. The invention is limited in that it teaches use of different microorganisms.
Payne et al. in U.S. Pat. No. 5,632,987 disclose a Bacillus thuringiensis toxin that is active against corn rootworm larvae. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Chilcott et al. in U.S. Pat. No. 5,645,831 disclose a Bacillus thuringiensis strain and metabolite that are active against corn rootworm. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Neyra et al. in U.S. Pat. No. 5,665,354 disclose a novel strain of Bacillus licheniformis that produces an antifungal agent and a use for the strain. The invention is limited in that it teaches use of a different microorganism.
Kubo in U.S. Pat. No. 5,667,779 discloses a fungi-inhibiting composition comprising Bacillus subtilis. The invention is limited in that it teaches use of a different microorganism.
Handelsman et al. in U.S. Pat. No. 5,695,982 discloses canavanine-resistant strains of Bacillus cereus. The invention is limited in that it teaches use of different microorganisms.
Neyra et al. in U.S. Pat. No. 5,697,186 disclose “flocculated bacterial cells” for use “as high-density crop inoculants” (Abstract). The reference is limited to the flocculation of one or more species of bacteria. Combination of bacteria with another type of microorganism is not taught by the reference. Nor, does it teach the combination of more than one bacterium will produce a greater response, only that the flocculation will allow the application of more than one bacterium for whatever reason.
O'Donnell in U.S. Pat. No. 5,702,701 discloses a process for treatment of soil and plants with a composition containing Bacillus laterosporus. The invention is limited in that it teaches use of a different microorganism.
Marrone et al. in U.S. Pat. No. 5,753,222 disclose an antibiotic-producing strain of Bacillus and methods of controlling plant diseases with it. The invention is limited in that it teaches use of a different microorganism.
Handelsman et al. in U.S. Pat. No. 5,852,054 disclose fungicidal toxins from a biocontrol bacterium. The invention is limited in that it teaches use of a different microorganism.
Marrone et al. in U.S. Pat. No. 5,869,042 disclose methods for controlling above-ground plant diseases. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Shetty in U.S. Pat. No. 5,882,641 discloses fruit pomice compositions and their uses. The invention is limited in that it teaches use of different microorganisms.
Howell in U.S. Pat. No. 5,882,915 discloses viridiol-deficient strains of Trichoderma virens and process for making and using biocontrol agents that contain them. The invention is limited in that it teaches use of different microorganisms.
Heins et al. in U.S. Pat. No. 5,906,818 disclose a Bacillus mycoides strain for controlling corn rootworm. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Smith et al. in U.S. Pat. No. 5,916,029 disclose a process for producing seeds coated with a microbial composition. The invention is limited in that it teaches use of different microorganisms.
Marrone et al. in U.S. Pat. No. 5,919,447 disclose a strain of Bacillus for controlling plant disease. The invention is limited in that it teaches use of a different microorganism.
Herrera-Estrella et al. in U.S. Pat. No. 5,922,603 disclose a method for obtaining strains of Trichoderma sp. The invention is limited in that it teaches use of a different microorganism.
Cook et al. in U.S. Pat. No. 5,972,689 disclose methods and compositions for control of root diseases. The invention is limited in that it teaches use of different microorganisms.
Eastin in U.S. Pat. No. 5,974,734 discloses solid-matrix priming of seeds with microorganisms. The invention is limited in that it teaches use of different microorganisms.
Handelsman et al. in U.S. Pat. No. 5,998,196 disclose a method for suppressing disease using a novel Bacillus cereus strain. The invention is limited in that it teaches use of a different microorganism.
Germida et al. in U.S. Pat. No. 6,015,553 disclose a Bacillus subtilis strain for controlling pests. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Logan et al. in U.S. Pat. No. 6,017,525 discloses a method of poultry house litter treatment. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Handelsman et al. in U.S. Pat. No. 6,030,610 disclose a novel strain of Bacillus cereus. The invention is limited in that it teaches use of a different microorganism.
Handelsman et al. in U.S. Pat. No. 6,033,659 disclose a novel strain of Bacillus cereus. The invention is limited in that it teaches use of a different microorganism.
Heins et al. in U.S. Pat. No. 6,103,228 discloses compositions and methods for controlling plant pests and a novel strain of Bacillus subtilis. The invention is limited in that it teaches use of a different microorganism.
Ocamb et al. in U.S. Pat. No. 6,133,196 disclose contacting conifer seeds with a culture of certain bacteria, drying the seeds, planting the seeds and then treating the plant growth medium with a certain ectomycorrhizal fungus (Hebeloma spp.). The reference does not teach treating seeds with a composition comprising a fungus and a bacterium.
Branly et al. in U.S. Pat. No. 6,232,270 discloses agricultural compositions containing bacteria. The invention is limited in that it teaches use of different microorganisms and a different process for their use. While the patent suggests that one bacterium that is useful within the disclosed invention is Bacillus amyloliquefaciens, it teaches that spores, cultures or suspensions of the bacterium must be combined with an agriculturally effective ingredient of a plant growth stunting agent or herbicide.
Moesinger in U.S. Pat. No. 6,326,016 discloses plant immunization compositions. The invention is limited in that it teaches use of different microorganisms and a different process for their use. While the patent suggests that one bacterium that can be used according to the disclosed invention is Bacillus amyloliquefaciens, and that one fungus that can be used according to the invention is Trichoderma spp., it teaches production of an extract by means of biotechnological fermentation process performed by only one microorganism. Furthermore, the patent teaches that high-temperature autoclaving (e.g., one hour at +120 degrees C.) and (optionally) filtering are further steps in preparation of the extract.
Background art is also disclosed in the non-patent literature. The StratSoy Research Database posted on the Web (apparently in 1996) a description of a research project entitled Interaction of Soybean Roots with Soilbome Pathogens and Nonpathogens. The project studied the use of Bacillus amyloliquefaciens B94 as a biocontrol agent for plant diseases. The invention is limited in that it teaches use of a single different microorganism.
An article entitled Seed's Coat of Many Microbes Wards Off Rot in Science Update section of the April, 1997, issue of Agricultural Research, disclosed the mixing of helpful fungi and bacteria to weaken or kill fungal pathogens that attack young corn sprouts. The applicant believes that the combination used was T. virens and Pseudomonas cepacia. This invention is limited for two reasons: (1) P. cepacia is implicated as causative in deaths in cystic fibrosis patients and, therefore, EPA registration would probably not be possible, and (2) P. cepacia is not a spore-forming bacterium and, therefore, the moisture content of the combination must be maintained at greater than about 17 percent. T. virens can be stored and applied at low moisture levels and does not remain in the spore state at the higher moisture levels, making its combination with P. cepacia impractical.
In an article authored by L. E. Datnoff et al. entitled Effect of Bacterial and Fungal Microorganisms to Colonize Tomato Roots, Improve Transplant Growth and Control Fusarium Crown and Root Rot that was posted on the Web in 2000 by the University of Florida Southwest Florida Research and Education Center, the authors discuss using a combination of an unidentified bacterial strain and Gliocladium virens to control Fusarium root rot. The invention is limited in that it teaches use of different microorganisms.
The U.S. Environmental Protection Agency Office of Pesticide Programs posted a Biopesticide Fact Sheet on Bacillus subtilis var. amyloliquefaciens strain FZB24 (006480) in May, 2000. The fact sheet noted that the strain was approved for use as a growth enhancer and disease suppressor. The invention is limited in that it does not teach the use of a fungal/bacterial combination.
No single reference and no combination of the references teach the invention disclosed herein. The background art does not teach combinations of microorganisms disclosed herein, combinations that provide a surprising consistency of performance in plant disease control.